Structural alterations in 70K with IGD mutations.

<p>Binding relative to no competitor of 1∶30,000 5C3 ascites (A) or 1∶50,000 7D5 ascites (B) to coated FN in the presence of increasing concentration of 70K (□), 70K I150/242A (▵), 70K I480/572A (▾), or 70K I150/242/480/572A (♦). Values are mean plus/minus standard deviation of 3 experiments.</p

N-³FNIII binds to FN^−/− fibroblasts.

<p>FN^−/− fibroblasts adherent to ¹FNIII-C EDA+-coated coverslips were provided 30 nM N-³FNIII or FN subunit (15 nM dimeric FN) in the presence of 200 nM LPA for 3 h, washed, fixed, and immunostained with the mAb 5C3 followed by rhodamine labeled secondary antibody. Bar, 10 µm.</p

70K with Ile-to-Ala mutations compete less well for binding of b-FUD to adsorbed FN.

<p>(A) Binding relative to no competitor of 0.3 nM b-FUD to coated FN in the presence of increasing concentrations of 70K (□), 70K I150/242A (▵), 70K I480/572A (▾), or 70K I150/242/480/572A (♦). (B) Binding relative to no competitor of 0.3 nM b-FUD to FN in the presence of increasing concentrations of human 70K (□) or rat 70K (▿). Values are mean plus/minus standard deviation of 3 experiments.</p

Diagram of FN and FN fragments and location of IGD motifs in 70K.

<p>(A) The EDA+, V89 splice variant subunit of FN is shown consisting of 12 FNI modules (ovals), two FNII modules (diamonds), and 16 FNIII modules (squares). Plasma FN lacks EDA and one subunit contains a variable region and the other subunit lacks it. Modules are numbered to facilitate naming recombinant proteins according to modular content. MSF is the N-terminus through the sequence encoded by the ¹FNIIIa exon and 10 intronic amino acids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030615#pone.0030615-Schor1" target="_blank">[6]</a>. FNI modules containing IGD motifs are indicated with an *. (B) Sequence of FUD with presumptive binding sites for FNI modules in bold and underlined and N- and C-terminal tails in lower case.</p

70K with Ile-to-Ala mutations bind to FN^−/− fibroblasts.

<p>(A) FN^−/− fibroblasts adherent to FN-coated coverslips were provided 40 nM FITC-70K, FITC-70K I150/242A, FITC-70K I480/572A, or FITC-70K I150/242/480/572A for 1 h before fluorescent microscopic imaging of the FITC fluorochrome. Results are representative of multiple fields of each condition examined in four separate experiments. Bar, 10 µm. (B) Western blot of cell lysates from cells provided FITC-70K (1), FITC-70K I150/242A (2), FITC-70K I480/572A (3), or FITC-70K I150/242/480/572A (4). The asterisk denotes the FITC-70K band. The Western blot was probed with rabbit anti-FITC followed by peroxidase-conjugated donkey anti-rabbit IgG.</p

Characterization of Molecules Binding to the 70K N‑Terminal Region of Fibronectin by IFAST Purification Coupled with Mass Spectrometry

Fibronectin (Fn) is a large glycoprotein present in plasma and extracellular matrix and is important for many processes. Within Fn the 70 kDa N-terminal region (70k-Fn) is involved in cell-mediated Fn assembly, a process that contributes to embryogenesis, development, and platelet thrombus formation. In addition, major human pathogens including <i>Staphlycoccus aureus</i> and <i>Streptococcus pyogenes</i> bind the 70k-Fn region by a novel form of protein–protein interaction called β-zipper formation, facilitating bacterial spread and colonization. Knowledge of blood plasma and platelet proteins that interact with 70k-Fn by β-zipper formation is incomplete. In the current study, we aimed to characterize these proteins through affinity purification. For this affinity purification, we used a novel purification technique termed immiscible filtration assisted by surface tension (IFAST). The foundation of this technology is immiscible phase filtration, using a magnet to draw paramagnetic particle (PMP)-bound analyte through an immiscible barrier (oil or organic solvent) that separates an aqueous sample from an aqueous eluting buffer. The immiscible barrier functions to remove unbound proteins via exclusion rather than dilutive washing used in traditional isolation methods. We identified 31 interactors from plasma, of which only seven were previously known to interact with Fn. Furthermore, five proteins were identified to interact with 70k-Fn from platelet lysate, of which one was previously known. These results demonstrate that IFAST offers advantages for proteomic studies of interacting molecules in that the technique requires small sample volumes, can be done with high enough throughput to sample multiple interaction conditions, and is amenable to exploratory mass spectrometric and confirmatory immuno-blotting read-outs

Characterization of Molecules Binding to the 70K N‑Terminal Region of Fibronectin by IFAST Purification Coupled with Mass Spectrometry

Fibronectin (Fn) is a large glycoprotein present in plasma and extracellular matrix and is important for many processes. Within Fn the 70 kDa N-terminal region (70k-Fn) is involved in cell-mediated Fn assembly, a process that contributes to embryogenesis, development, and platelet thrombus formation. In addition, major human pathogens including <i>Staphlycoccus aureus</i> and <i>Streptococcus pyogenes</i> bind the 70k-Fn region by a novel form of protein–protein interaction called β-zipper formation, facilitating bacterial spread and colonization. Knowledge of blood plasma and platelet proteins that interact with 70k-Fn by β-zipper formation is incomplete. In the current study, we aimed to characterize these proteins through affinity purification. For this affinity purification, we used a novel purification technique termed immiscible filtration assisted by surface tension (IFAST). The foundation of this technology is immiscible phase filtration, using a magnet to draw paramagnetic particle (PMP)-bound analyte through an immiscible barrier (oil or organic solvent) that separates an aqueous sample from an aqueous eluting buffer. The immiscible barrier functions to remove unbound proteins via exclusion rather than dilutive washing used in traditional isolation methods. We identified 31 interactors from plasma, of which only seven were previously known to interact with Fn. Furthermore, five proteins were identified to interact with 70k-Fn from platelet lysate, of which one was previously known. These results demonstrate that IFAST offers advantages for proteomic studies of interacting molecules in that the technique requires small sample volumes, can be done with high enough throughput to sample multiple interaction conditions, and is amenable to exploratory mass spectrometric and confirmatory immuno-blotting read-outs